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    • Ligand independent constitutively active variants have also

    2020-07-02

    Ligand-independent, constitutively active variants have also been found in receptors with associated tyrosine kinases. In approximately 60% of patients harboring inflammatory hepatocellular adenomas (IHCAs), these adenomas have been caused by small in-frame deletions in the cytokine-binding domain of the IL-6 signal transducer chain gp130, Senexin A mg resulting in permanent intracellular receptor dimerization and activation 75, 76, 77. Although most mutations affect the extracellular domains of cytokine receptors, frameshift and nonsense mutations in the EPO receptor have resulted in gain-of-function receptors with cytoplasmic truncations along with the loss of the C-terminal domain with docking sites for positive or negative regulators of EPO receptor (EPOR) signaling 78, 79. Furthermore, a constitutively active semi-synthetic IL-7R (C7R) was engineered and introduced into CAR T Senexin A mg [80]. Coexpression of the constitutively active IL-7R with a tumor-directed CAR (GD2-CAR) in T cells was shown to increase T cell proliferation, survival, and antitumor activity in vitro, and was also effective in reducing metastatic neuroblastoma and orthotopic glioblastoma in mouse xenograft models [80]. This synthetic IL-7R was developed from a natural variant found in acute lymphoblastic leukemia (ALL) patients, which forms homodimers due to cysteine and/or proline insertions in the transmembrane domain, and constitutively signals without the need for IL-7 stimulation or the γ chain receptor subunit [81]. Moreover, constitutively active IL-7R phenocopied results obtained with T cells genetically modified to secrete IL-7 82, 83. From another angle, constitutively active synthetic receptor variants have been generated by replacing the extracellular domain with other domains such as leucine zippers (coiled-coil domains) or by generating deletions within the receptor stalk regions of a variety of cytokine receptors including EGFR, growth hormone (GH) receptor, and gp130 family members 84, 85, 86, 87, 88.
    Synthetic Cytokine/Cytokine Receptor Systems for Immunotherapies Aside from the discovery of naturally occurring constitutively active cytokine receptors, significant progress has been made in the generation of switchable synthetic cytokine receptors, including fully synthetic cytokine systems featuring combinations of synthetic ligands and synthetic receptors. As early as 1993 a synthetic receptor/ligand system based on cell permeable dimerized immunophilin ligands was generated [89]. Switchable membrane-bound chimeric cytokine receptors composed of the extracellular domains of the EPOR and intracellular domains of gp130 were described in the 1990s, demonstrating the principle of extra- and intracellular domain shuffling that have been transferred to many receptors over the last two decades 90, 91, 92. Recently, thermoresponsive elastin-like polypeptides (ELPs) have been fused to the C terminus of EGFR. In this model, before thermal stimulation, the EGFR was found in the ‘off’ activation state; however, short heating in the range of 25–42°C gradually induced ELP-phase separation, aggregation of EGFR–ELP fusion proteins, and induction of signal transduction, as demonstrated by ERK phosphorylation, in cultured 293T cells in the absence of natural EGFR ligands [93]. Of note, optogenetics approaches have also been adopted to facilitate and induce cytokine signaling (Box 1). Alternatively, receptors may be customized to fulfill novel tasks, as has been demonstrated for the modular extracellular sensor architecture (MESA) receptors that enable cells to sense vascular endothelial growth factor (VEGF) and, in response, secrete IL-2 [94]. MESA receptors are composed of a target chain (TC) and a protease chain (PC). Both contain an extracellular domain (ECD) recognizing a ligand of choice, a scaffold region connecting the ligand sensing ECD to the membrane, followed by a transmembrane domain, a flexible intracellular linker domain and a sequestered transcription factor on the target chain, or a site-specific protease on the adjacent protease chain. In this study, VEGF induced dimerization of anti-VEGF scFvs fused to a MESA scaffold [95], leading to intracellular trans-cleavage of the target chain by the protease chain in cultured human T cells. This in turn, resulted in the release of a sequestered and readily programmable transcription factor based on the CRISPR–Cas9 genome editing system. To generate this transcription factor, catalytically inactive Cas9 was fused to the VP64 transcriptional activation domain. Release of this transcription factor upon VEGF stimulation of cells cotransfected with the synthetic MESA receptor and a guide RNA directing the transcription factor to the IL2 locus, resulted in IL-2 expression 94, 95. This system offers a general strategy for generating programmable cellular functions and might even be combined with immunocytokine neoleukin expression.